The long-term goals of this project are to 1) understand the role of mitochondria in ischemia-reperfusion njury and cardioprotection ; 2) to understand the role of altered ion homeostasis and altered metabolism in ischemia-reperfusion and cardioprotection and 3) to understand changes in cytosolic and mitochondrial signaling involved in cardioprotection and cell death. It is proposed that ischemic preconditioning (PC) initiates signaling that converges on mitochondria and results in cardioprotection. It has been proposed that cells die following ischemia and reperfusion because of a rise in mitochondrial calcium which leads to activation of the mitochondrial permeability transition pore (MPT). The increase in mitochondrial calcium is proposed to occur via calcium uptake by the mitochondrial calcium uniporter. The composition of the MPT is unknown, but cyclophilin D has been shown to be a regulator of the MPT. We therefore attempted to gain information on the MPT by examining the physiological role of cyclophilin D. Isolated mitochondria from mice deficient in cyclophilin D (CypD-/-) are less sensitive to Ca2+-induced opening of the mitochondrial permeability transition (MPT) in vitro. Thus, the lack of CypD enables heart mitochondria to take up more Ca2+ before undergoing the MPT. We hypothesize that the MPT serves as a Ca2+-safety valve that can open to release excess Ca2+, but not necessarily result in death. If the MPT is blocked in CypD-/- mice, we hypothesize that matrix Ca2+ (Ca2+m) would be higher in CypD-/- mice compared to WT and this would activate Ca2+-sensitive NADH dehydrogenases (e.g., pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH)), which would in turn, alter oxidative metabolism and increase oxygen consumption. Consistent with this, we found altered expression levels of PDH E1 subunit and the alpha-KGDH E2 subunit in CypD-/- hearts using 2D DIGE proteomics. Therefore, these results demonstrate that the loss of a MPT component, CypD, results in physiological flux changes in the Krebs cycle and oxidative metabolism that are consistent with increased mitochondrial Ca2+. Thus, mice lacking cyclophilin D (CypD-/-), a mitochondrial chaperone protein, have altered cardiac metabolism. As acetylation has been shown to regulate metabolism, we tested whether changes in protein acetylation might play a role in these metabolic changes in CypD-/- hearts. To identify changes in lysine-acetylated proteins and map acetylation sites following ablation of CypD, we subjected tryptic digests of isolated cardiac mitochondria from WT and CypD-/- mice to immunoprecipitation using agarose beads coupled to anti-acetyl lysine antibodies followed by mass spectrometry. We used label-free analysis for the relative quantification of the 875 common peptides that were acetylated in WT and CypD-/- samples and found 11 peptides (10 proteins) decreased and 96 peptides (48 proteins) increased in the CypD-/- samples. We found increased acetylation of proteins in fatty acid oxidation and branched-chain amino acid metabolism. To evaluate whether this increase in acetylation might play a role in the inhibition of fatty acid oxidation that was previously reported in CypD-/- hearts, we measured the activity of L-3-hydroxyacyl-CoA dehydrogenase (LCHAD), which was acetylated in the CypD-/- hearts. Consistent with the hypothesis, LCHAD activity was inhibited by approximately 50% compared to the WT mitochondria. These results implicate a role for CypD in modulating protein acetylation. Taken together, these results suggest that ablation of CypD leads to changes in the mitochondrial acetylome, which may contribute to altered mitochondrial metabolism in CypD-/- mice. We were also interested in examining the role of calcium uptake by mitochondrial calcium uniporter (MCU) in regulating cell death and metabolism. In collaboration with Dr. Toren Finkel, we characterize a mouse model that lacks expression of the recently discovered mitochondrial calcium uniporter (MCU). Mitochondria derived from MCU(-/-) mice have no apparent capacity to rapidly uptake calcium. Whereas basal metabolism seems unaffected, the skeletal muscle of MCU(-/-) mice exhibited alterations in the phosphorylation and activity of pyruvate dehydrogenase. In addition, MCU(-/-) mice exhibited marked impairment in their ability to perform strenuous work. We further show that mitochondria from MCU(-/-) mice lacked evidence for calcium-induced permeability transition pore (PTP) opening. Surprisingly, the lack of PTP opening does not seem to protect MCU(-/-) cells and tissues from cell death. Following global I/R in a Langendorff perfused heart model infarct size was indistinguishable between WT hearts and hearts from MCU-/- mice. To further complicate the picture, cyclosporine A, an inhibitor of the mPTP, reduced infarct size in WT hearts, but not in MCU-/- hearts.